Understand the Regulation of Learning and Memory Formation from a Molecular Prospective

BPJ_112_6.c1.inddWhen most people talk about calcium (Ca2+), they think about building bones and muscle contraction. In fact, calcium is also essential for learning and memory formation. Molecular basis for learning and memory formation has aroused attention since 1980s.  So what does calcium do with learning and memory formation? The calcium-modulating protein calmodulin (CaM) coordinates the activation of a family of Ca2+-regulated proteins, which are crucial for synaptic plasticity associated with learning and memory in neurons. These proteins include neurogranin (Ng) and CaM-dependent kinase II (CaMKII). In a resting cell, CaM is mostly reserved by Ng and free of Ca2+, whereas in a stimulated cell, CaM is able to bind Ca2+ and activate CaMII, which plays a pivotal role in learning and memory formation for both long-term potentiation and mechanisms for the modulation of synaptic efficacy.

The cover image for the March 28 issue of the Biophysical Journal shows the crystal structure of CaM-CaMKII peptide and the structure of CaM-Ng from coarse-grained molecular simulations. CaM molecules are ribbons in silver, calcium ions are represented by yellow beads, CaMKII peptide is in green surface representation, and Ng peptide is in red surface representation. One CaM-Ng peptide complex is near, where the Ng is aligned with a “pry” (pink); the other is far, indicating rich level of Ng. The images were rendered using the software Visual Molecular Dynamics developed by University of Illinois at Urbana-Champaign with the built-in Tachyon ray tracer. The illustration of a neuron in hippocampus is taken from Shelley Halpain, UC San Diego. Dendrites are green, dendritic spines red, and DNA (in cell nucleus) blue. The illustration of a human brain contains red dots to indicate active parts of the cerebral cortex.

Our computational study provides the very first detailed description at atomistic level ofhow binding of CaM with two distinct targets, Ng and CaMKII, influences the release of Ca2+ from CaM, as a molecular underpinning of CaM-dependent Ca2+ signaling in neurons. We believe this study bridges the molecular regulations in atomistic detail and the understanding of cellular process cascade of learning and memory formation.

– Pengzhi Zhang, Swarnendu Tripathi, Hoa Trinh, Margaret S. Cheung

Biophysics and Bleeding Disorders

March is Bleeding Disorders Awareness Month in the US. More than three million Americans who have hemophilia, von Willebrand disease, and other rare bleeding disorders. These conditions prevent blood from clotting the way it should, which can lead to prolonged bleeding after injury, surgery, or physical trauma. We spoke with Biophysical Society member Valerie Tutwiler, an American Heart Association graduate research fellow in the lab of John Weisel at the University of Pennsylvania, about her hemostasis and thrombosis research.

What is the connection between your research and bleeding disorders?

BPJ_112_4.c1.indd

This recent cover of Biophysical Journal shows Tutwiler, Wang, Litvinov, Weisel, and Shenoy’s image of a colorized scanning electron microscope image of a coronary artery thrombus extracted from a heart attack patient.

Blood clotting or hemostasis is the process that stems bleeding. On one hand if you have insufficient clotting this can result in prolonged bleeding, on the other hand a hypercoagulable state can result in thrombosis. Thrombi can result in the obstruction of blood flow, which can cause heart attacks and strokes. My thesis research pertains largely to studying one portion of the coagulation process blood clot contraction, or the volume shrinkage of the clot, which has been implicated to play a role in hemostasis and the restoration of blood flow past otherwise obstructive thrombi.

Why is your research important to those concerned about bleeding disorders?

While there is much known about the various aspects of blood clotting relatively little is known about the process of clot contraction despite the clinical implications of its importance in the formation of a strong hemostatic seal and the restoration of blood flow past otherwise obstructive thrombi. The study of clot contraction is a highly interdisciplinary problem and as a result can be of interest to researchers from many different fields. Platelets are active contractile cells, which interact with an extracellular matrix of fibrin, a naturally occurring polymer with unique mechanical properties. The fibrin matrix can be imbedded with other blood cells, such as red blood cells, as well. From a biophysical standpoint the mechanisms of clot contraction have not been well understood. To better elucidate this process, we performed a systematic study on how the molecular and cellular composition of the blood influences the rate and extent of clot contraction along with the mechanical properties of the contracting clot using a novel application of an optical tracking system.

Additionally, to further explore the mechanical nature of the clot contraction process we developed a mathematical model that couples active platelets with a passive viscoelastic matrix made up of fibrin and red blood cells. The model predicts the process of clot contraction and explains some of the experimental observations of clot size, structure and mechanical forces. Interestingly, we found that clot contraction is altered in thrombotic states such as ischemic stroke patients. Collectively, these findings show that the study of clot contraction has the potential to inform the development of diagnostics and therapeutics.

How did you get into this area of research?

Since beginning research I have been interested in applying engineering techniques to answer biological questions. I became interested in hemostasis and thrombosis research while completing my first co-op experience in undergrad.

How long have you been working on it?

I began doing hemotology research during my undergraduate career. However, I started studying clot contraction specifically when I started my PhD research.

Do you receive public funding for this work? If so, from what agency?

I am currently funded by the American Heart Association as a pre-doctoral fellow, although we also receive funding from the National Institute of Health and National Science Foundation.

Have you had any surprise findings thus far?

We were surprised to find such a striking decrease in the extent of clot contraction in ischemic stroke patients compared to healthy subjects. Correlations with stroke severity suggest that clot contraction may be a potential pathogenic factor in ischemic stroke. These findings have led us to expand our study to other pathological conditions as well.

What is particularly interesting about the work from the perspective of other researchers?

Due to the conservation of the basic principles of contractile proteins and motility, the information learned from the development of a mathematical model of active contractile cells interacting with a viscoelastic matrix can be applied to a variety of different processes.

What is particularly interesting about the work from the perspective of the public?

Bleeding and thrombotic conditions remain leading causes of death and disability worldwide. Gaining a more thorough understanding of the processes involved in hemostasis and thrombosis will lead to the development of more effective diagnostic tools and more targeted therapeutics.

PhosphoHero is in Charge of Neurofilaments’ Order

BPJ_112_5.c1.inddGels are neither solids nor liquids but rather a network of deformable and crosslinked polymers. Therefore, it is not surprising that the mechanical properties of synthetic gels are controlled by the degree of cross-linking, achieved, for example, by photopolymerization or the addition of chemical agents. One of the best examples of mechanically supporting bio-gels is the cytoskeleton, where crosslinked polymers (actin, microtubules and intermediate filaments) form a viscoelastic network. For microtubules and actin networks, analogues biological cross-linkers (associated proteins) have been identified. Nonetheless, in some important cases, the biophysical crosslinking mechanism or the existence of associated crosslinking proteins have not been identified.

Neurofilaments (NF) are neuronal specific intermediate filaments that form spaced filamentous networks in the long axon projections. Each neurofilament resembles a bottlebrush: a semi-flexible filament decorated with protruding floppy (intrinsically disordered) long carboxyl terminal tails. The tails engage in extensive crosslinking interactions, which have been the focus of many studies.

In addition to their lack of secondary rigid structure, NF tails contain many “phosphorylation sites”. These sites are specific amino-acid sequences recognized by enzymes that can add or remove charged phosphate groups, known as phosphorylation and dephosphorylation, respectively.

Our cover image for the March 14 issue of the Biophysical Journal illustrates an NF gel made of well aligned bottlebrushes at the front, and un-oriented ones at the back. The superhero (PhosphoHero) artistically illustrates the roles of NF phosphorylation. On the one hand, PhosphoHero increases the cross-linking between the filaments via the generation of ionic bridging between opposite charged residues. This in turn aligns the red filaments in nematic liquid crystalline order, as depicted by the crossed polarized NF hydrogel microscopy in the background. On the other hand, phosphorylation also increases the tails’ net negative charge, and consequently its compression response. Thus, phosphorylation acts as a regulatory knob to control the structure, orientation and mechanical properties of the cellular scaffold, the cytoskeleton.

Future studies into the role of intrinsically disordered proteins, and in particular their tunable phosphorylation states and their role in long-range alignment should be full of further surprises. Intrinsically disordered proteins were evolutionally selected to hold functional, although sometimes atypical properties, characteristic to superheroes.

The cover was hand-drawn and then digitally colored in Photoshop by Eliran Malka.

– Eti Malka-Gibor, Micha Kornreich, Adi Laser-Azogui, Ofer Doron, Irena Zingerman-Koladko, Jan Harapin, Ohad Medalia, Roy Beck

How to Write a Biophysics Article Worthy of Publication: Part 3- From Submission to Acceptance

William O. Hancock
Pennsylvania State University
Member, Biophysical Society Publications Committee
wohbio@engr.psu.edu

The first part of this series covered writing a first draft of a manuscript, and the second part covered the honing and polishing needed to bring the manuscript to the point where it is ready to submit to a journal.  The topic of this final article is navigating the process of submitting, revising, and getting your manuscript accepted for publication.

Choosing a journal

Because this piece is written with the Biophysical Journal in mind, your manuscript has hopefully developed into an appropriate submission to that journal.  From the journal website:

The mission of Biophysical Journal (BJ) is to publish the highest quality work that elucidates important biological, chemical, or physical mechanisms and provides quantitative insight into fundamental problems at the molecular, cellular, and systems, and whole-organism levels. Articles published in the Journal should be of general interest to quantitative biologists, regardless of their research specialty.

If your manuscript has evolved away from this definition, then you may want to choose another journal.  A good guide is to consider what journals are commonly read by colleagues in your field and fields relevant to your work.  Don’t be overly swayed by impact factors, and avoid predatory journals.  Consider the makeup of the Editorial Board who will be deciding on whether your manuscript is sent to review, and consider the business model of the journal.  Society-based journals (such as Biophysical Journal) carry the weight of the Society, usually have a history, and are generally run by scientists for scientists.

Before submitting your manuscript (and during the process of writing drafts and polishing your figures), consult the Guide for Authors and follow formatting, word count, and figure guidelines.  This will speed the submission and review of your manuscript, it increases the chance of acceptance, and it will save you time during later revision steps.

Most journals accept pre-submission inquiries to assess the suitability of the manuscript for the journal (and some journals require them).  This process involves sending your title and abstract together with a short letter to the editor, and it saves time for everyone involved.

Navigating the review process
picture-3The process of submitting a manuscript involves a number of decision points that are shown in the figure at right.  Upon initial submission, an editor will decide if the manuscript should be reviewed or be rejected (triaged) at this initial submission stage.  Considerations include suitability of the topic for the journal, novelty of the work, completeness of the work, and perceived impact.  Although it can be discouraging, this initial triage is another important time saver for everyone involved.  Avoiding rejection at this juncture can be helped by a pre-submission inquiry to determine suitability, and by a convincing cover letter.

Cover letter

One element that is sometimes underappreciated by authors is the cover letter, which provides the author a platform to persuade the editor of the importance of the work and its suitability for the journal.  The editor will generally be asking two questions:  (1) Is this work significant?  (2) Do the results justify the conclusions?  In the letter, it is important to distill the key findings into a few sentences.  However, more importantly, you want to place the work in the larger context of your field, and of the larger field of biophysics, cell biology, structural biology, or whatever your specialty may be.  This larger perspective is what the editor is thinking about — what is the impact of this manuscript, and will publishing it advance the mission of the journal?  Therefore, it can help to point out important recently published work by yourself and others that relates to the manuscript.  It is also good to remind the editor of the larger impact of the work on medicine, basic science, or technology.  Some of this persuasion means plucking text from the Introduction or Discussion of the manuscript, but it also requires stepping out to more of a 30,000 foot perspective and persuading the editor in a way not unlike a grant application.  Be specific and persuasive without being grandiose.

What makes an effective review?

Now that your manuscript has made it to peer review, it will be read by two or more reviewers who are considered experts in the subject of your manuscript.  The primary goal of the reviewers is to ask:  Do the results justify the conclusions?  A good review should provide substantive feedback that enables the editor to make an informed decision on the manuscript and the authors to revise and improve the manuscript.  Reviews generally begin with a brief summary of the findings and their relevance to the field, and may include the following:

  • A critical evaluation of the experiments, highlighting any flaws in experimental design, questionable interpretation of data, and any internal consistencies.
  • Highlighting previously published work (with references) that either contradict the work or may make the current experiments redundant.
  • Reasonable requests for further experiments, particularly control experiments but also obvious (important) experiments that the authors may have neglected.
  • Request for further analysis, reanalysis, or alternative presentation of experimental data, including adding or clarifying statistics.
  • A critique of the text and figures highlighting areas of confusion, excessive verbosity, or flawed logic.

A good review will be civil, will avoid vague complaints, and will not harp unnecessarily on small details that may not be related to the principal point of the manuscript.  The authors and editor are helped most by specificity and forthrightness in the evaluation of the manuscript.

Revising and responding to reviews

When the editor receives the reviews back, they then make a decision either to accept the manuscript as is (which is rare), reject the manuscript, or ask for major or minor revisions.  At this point, the author has to make a decision. Rejections can be appealed in select cases, but this avenue should be used sparingly and should have strong justification.  If the appeal is denied, then the authors should incorporate suggestions from reviewers before resubmitting to another journal, because it is likely that other reviewers will have the same complaints.

If minor revisions are requested, the authors can generally address the comments by editing the text, improving the figures, or making other modifications that don’t take much time.  In this case, the authors should attend to these tasks immediately and resubmit the revision.  In the case of major revisions, the authors have other decisions to make.  In some cases, the revisions and additional experiments requested are so extensive that it essentially requires rewriting the manuscript.  Depending on constraints, the best avenue may be to make minor modifications and submit it to a more specialized or lower profile journal.  If the decision is to revise and resubmit, then the authors must make a battle plan that involves some combination of further experiments, reanalysis of data, and revising the text and figures.  Often a limit of 90 or 120 days for resubmission is given (though deadlines can usually be extended by a reasonable request); this timeline provides a scale of the amount of new work that is expected.

When resubmitting a manuscript, the authors should also submit both a marked copy that highlights changes, and a point-by-point response to the reviewer comments.  It is expected that authors make a good faith effort to make edits and carry out further analysis and experiments.  A letter that tries to simply rebut every suggested experiment will not generate good will with the editor or reviewers.  That being said, it is reasonable to carry out some of the experiments suggested by reviewers and rebut suggested experiments that are onerous or extraneous.  Editors and reviewers will be more inclined to accept an explanation for not doing an experiment if you have followed their directive on other suggested work.  In some cases, data addressing a reviewer concern can be presented in the response to reviewers letter and not included in the text of the revised manuscript.

Upon resubmission, the editor may decide to accept the manuscript, or they may send it back out for review. At this point, the manuscript will be re-evaluated by one or more of the original reviewers.  In some cases, a new reviewer may be added to address a particular aspect of the manuscript.  If a major revision is requested and the authors have not carried out the requested experiments or sufficiently revised the work, the manuscript may be rejected at this point.  If the revisions were extensive and the reviewers still have complaints, then the manuscript may be sent back to the author for another round of revisions.  While this action is necessary in some cases, the extra work and time can be avoided by authors responding fully to critiques on their first revision and by reviewers detailing all of their concerns on their initial review and abstaining from making new critiques of aspects of the manuscript that were not commented on during the first round.

Publishing your paper

Hopefully this process will culminate with your manuscript being accepted for publication.  Congratulations!  But before you can move on to your next paper, there are a number of details to take care of.  First, it is imperative that the final revision that was submitted is error free.  It is worth taking the time now to be sure that the version that the journal has in hand has all figure numbers correct, all references in order, and other small details in place.  This is also the last time you will be able to edit the Supplemental Information, so be sure that document is properly formatted and is complete.  You will be sent page proofs for final checking, but it is best to have everything ironed out before the manuscript goes to proof stage, so that the final stage only involves checking for typesetting errors, figure placement, and related small details.

Over this three-part series, we have gone from data in a lab notebook to a published paper.  This process takes a lot of work, and although it gets easier the more you do it, publishing a paper is always a considerable effort.  However, peer-reviewed publications are the currency of science, and so the effort is necessary and worth it, and reaching this milestone is cause for celebration.  And, after the celebration dies down, then get back to the lab and do it again…

Acknowledgements

The author thanks Beth Staehle for assistance and advice, Olaf Anderson for many of the ideas that went into this work, and members of the Biophysical Society Publications Committee for many helpful suggestions.  He also thanks his mentors Joe Howard and Al Gordon, as well as his 8th grade grammar teacher, Jim Ernst, for teaching him how to write.  W.O.H. is supported by the NIGMS.

Helpful online resources

In addition to the references presented in Part 2 of this series, there are a number of more general resources online to help improve your scientific communication.

https://cgi.duke.edu/web/sciwriting/

  • An excellent online writing resource with tutorials that focus on science writing fundamentals

http://www.nature.com/scitable/ebooks/english-communication-for-scientists-14053993/writing-scientific-papers-14239285

  • Helpful eBook on writing scientific papers from Nature Education

http://www.ncbi.nlm.nih.gov/books/NBK988/

  • A useful style guide, particularly for questions on grammar

http://www.americanscientist.org/issues/pub/the-science-of-scientific-writing

  1. Gopen and J. Swan. The Science of Scientific Writing. American Scientist, November-December 1990.
  • An in-depth article that focuses on the readers’ perspective and breaks down sentence and paragraph structure for maximum communication

Books:

Michael Alley, The Craft of Scientific Writing, 3rd Edition, Springer, 1995.

Michael Jay Katz, From Research to Manuscript:  A Guide to Scientific Writing.  Springer Netherlands, 2009.

Interested in learning more about writing a good biophysics article?  Want a chance to ask questions?  Dr. Hancock will present a webinar on this subject today, March 10, at 1:00 PM ET.  Register at http://www.biophysics.org/Education/Webinars

 

 

 

The Science Behind the Image Contest Winners: Fluorescent Muscles in Living C. elegans

The BPS Art of Science Image Contest took place again this year, during the 61st Annual Meeting in New Orleans. The image that won third place was submitted by Ryan Littlefield, assistant professor, Department of Biology, University of South Alabama. Littlefield took some time to provide information about the image and the science it represents.

ImageJ=1.47v

How did you compose this image?

Usually these C. elegans worms move around quite vigorously.  I added muscimol to prevent muscle contraction.  I picked three different types of worms that appear red only, green only, and red and green (which appear mostly yellow) and mixed them together on a thin pad of agarose.  The worms in the image all happened to clump together, resulting in a nice demonstration of the different color patterns.  I collected Z-stacks for each of the fields of view on an Andor spinning disk confocal microscope.  Using ImageJ software, I then made projections for the images that included the body wall muscle of the worms and pair-wise stitching of about six different projections.

What do you love about this image? 

The juxtaposition of all three types of transgenic worms being next to each other was very striking. The image includes all the different regions of the worms in various orientations, shows many of the different muscle types in these worms, and shows how the muscle cells fit together.

What do you want viewers to see or think about when they view this image?

The striated myofibrils in these worms are beautifully organized along their lengths, and it naturally raises the question of how this organization is achieved. In addition, the different muscle types show the viewer that there is a lot of diversity along the length of these 1mm worms.

How does this image reflect your scientific research?

I am interested in how actin and myosin become organized into functional, contractile bundles. In particular, I am interested in how actin filament lengths are specified in striated muscle.  In these worms, I modified an isoform of muscle myosin and tropomodulin with fluorescent proteins (mCherry and GFP, respectively) to determine how thin filaments are organized within these muscles.

What are some real-world applications of your research?

Both the uniformity of and specific lengths of actin filaments are important components of muscle physiology. Misregulation of actin filament lengths may be important factor in diseases including cardio- and skeletal and myopathies.  In addition, muscle damage from extended spaceflight, sarcopenia from aging, and acute muscle injuries may be slowed or prevented by interventions that prevent actin filament lengths from changing.

How does your research apply to those who are not working in your specific field?

Striated myofibrils are a dramatic example of a dynamic, self-organizing biological system that is attuned to a specific function (contraction).  Similar mechanisms for functional self-organization may also be used for other contractile actomyosin bundles, such as stress fibers and contractile fibers in smooth muscle, and for other dynamic cytoskeletal systems, such as flagella and microtubules in the mitotic spindle.

How to Write a Biophysics Article Worthy of Publication: Part 2- From First Draft to Final Draft

William O. Hancock
Pennsylvania State University
Member, Biophysical Society Publications Committee
wohbio@engr.psu.edu

“I have never thought of myself as a good writer. But I’m one of the world’s great rewriters.”

James A. Michener

Part 1 of this series covered the task of transforming data in your lab notebook and thoughts in your head into a first full draft of your manuscript.  The next task is to convert this rough draft into a polished manuscript that you can publish and be proud of.  This process requires streamlining your message, honing your logic, and achieving clarity and conciseness in your prose.  You will likely work through a number of drafts, and revising will probably take significantly longer than writing your first draft, but this effort is essential to create a publication-quality manuscript.  Here I detail the key steps of this process.

Revisit your story

Ask yourself: Have I achieved my goal of presenting a compelling story for a specific audience?  Don’t worry that the topic may have drifted far from where you started when you first sat down to write.  Your story should be presented as a logical progression of experiments that build upon one another to convince the reader of your main point.  Hence, consider the logic and try to think from the point of view of the reader.  You may decide at this point to significantly re-sequence your figures and the subsections that make up the Results section. Don’t be afraid of “major surgery” as moving big pieces is easy, and a smooth and logical flow is essential.  You may also realize that one (or more) figures contributes little to the essential narrative and can therefore be deleted or demoted to Supplemental Information.  If you find yourself holding on too tightly to your hard-won text or plots, keep in mind the following quotes:

“In writing, you must kill all your darlings.”
William Faulkner

“The more you leave out, the more you highlight what you leave in.”
Henry Green

Before setting out to revise your first draft, consult the Guide for Authors for the journal you are targeting, and follow word count, formatting, and figure guidelines.  Doing this in advance will save you a lot of later work during the final journal submission steps.

Hone your writing

Now it’s time to pick apart your text and to tighten up your writing to maximize the clarity and impact of your message.  There are many good writing resources available, but here I’ll highlight some key points:

  • Each paragraph should make a single point that is ideally presented in the first sentence (the topic sentence). The last sentence of a paragraph should link it to the topic of the next paragraph. Some people write outlines with the first sentence of each paragraph written out and write a draft from there.  That is a good practice, and when revising you can do this retroactively to track the overall organization of the manuscript.
  • When writing, strive to be clear as well as terse. Don’t use extra words (instead of “at this point in time” use “now”; instead of “a large majority of” use “most”).  Don’t use pompous language (replace “utilize” with “use”; avoid the phrase “needless to say”).  Never use the word “believe” in scientific writing.  Watch out for the word “prove”; instead use “suggest,” “indicate,” or “are consistent with.”  It is also best to use the active voice when writing.
  • Avoid lab jargon. Consider renaming your constructs or methods from the terms that you routinely use in the lab to more specific terms that readers can understand and remember, and that are consistent with previous use in the literature.
  • Minimize acronyms because, although they save space, they are one more thing the reader must keep in their mind. So, err on the side of clarity and inclusiveness (broad readership), and when possible write them out.

Think about your audience

As you hone your writing, maintain a focus on educating and informing your reader — try to make it easy for them.  In the Introduction, think of the essential background material they need to know in order to understand your study.  In the Results, clearly explain what the data do and do not say and emphasize the most important data.  In the Discussion, clearly explain the implications (as well as the limits) of the work and how it relates to what has been done before.

One way to help your reader understand and remember your message is through repetition.  There is a useful old saying:  “Tell ‘em what you’re gonna’ tell ‘em … tell ‘em’ … tell em’ what you told ‘em.”  In the structure of a scientific manuscript this means that in the last paragraph of the Introduction you need to preview the results, in the Results you need to clearly present the findings, and in the Discussion you need to reiterate and expand on the findings.

A second strategy is to build up from the highly believable (established or simple) to the less believable (new) (Senturia, 2003).  At the level of the entire manuscript, this means the Introduction sets up what is known (believable) and the Discussion allows for your speculation and making links to other work (less believable).  This idea also applies to the Results — you should generally start with the simplest results and build up to the most novel and surprising.  You are establishing the readers’ (and reviewers’) trust and providing them with a firm foundation on which to interpret your most exciting findings.

A final point is: Don’t overestimate how much information a reader can absorb and remember.  There is always a temptation to present all of your data and make as many points as possible.  However, more data can paradoxically reduce the impact of a paper by diluting the message.  If your results revolve around a single central point of the paper, you have a good chance of having the reader come away with that point and remember it hours, days, or weeks later.  If you are trying to make three loosely related points, your odds go way down.  Hence, consider cutting and demoting some data to Supplemental Information — or in extreme cases — even splitting a paper that is bursting at its seams into two.

Make your figures beautiful

Revisit your figures to ensure that they are informative and uncluttered, and that they connect tightly to the text in the Results section.  Every panel of every figure should be referenced in the text (if you don’t reference a panel, cut it).  Think of the key point you want to get across in each panel, and use that to guide precisely how you want to plot your data.  Can you remove non-essential data? Change symbols or add labels or lines to emphasize the key point?  A few points to remember:

  • Make your symbols sufficiently large to see, and make them consistent throughout the manuscript. Are the axes clearly labeled with sufficiently large fonts (keep in mind that figures may be reduced in size by the journal)?  Consider the range — ideally start with zero at each origin and choose a maximum value on each axis that highlights the important variation of the data and also shows any plateau effect.
  • Are you plotting the data in the optimal way? Bar plots are notorious; not only do they distill a distribution down to a single mean but, because of equal spacing on the x-axis, they can obscure important time and concentration dependencies.  For measurements that depend on a quantitative variable, consider an x-y scatter plot.  Or, instead of presenting a simple mean or a “bar and whiskers” plot, consider using a “Bean Plot” for moderate N values to show every individual measurement, or a “Violin Plot” for high N values to show their distribution (Weissgerber et al., 2015; Spitzer et al., 2014).
  • All images should have scale bars that are labeled with units on the figure or in the figure legend. Ask yourself whether you should crop to emphasize the key element in the figure.  Avoid nonlinear contrast enhancement in images, gels, and blots.
  • Consider what data to put into Supplemental Information. Are there raw data that can be presented that are informative?  Are there key control experiments that are important but don’t fit particularly well in the main results?  The phrase “data not shown” should be avoided if possible (some journals even prohibit it), and the data instead should be included as Supplemental Data.  However, avoid the temptation of putting extra data into Supplemental just because you did the experiments and you want to put it somewhere.

image-2

Honing specific sections

Introduction: 

Does your first paragraph set up the paper?  It should not be overly general background information; instead it should focus the questions being addressed.  Is referencing correct throughout the Introduction?  Apart from the most general statements, any time you state that something is “known” or you are stating a “fact,” you need to reference it (using original research articles rather than reviews where possible).  Avoid excessive self-referencing.  Avoid long strings of references; a general rule of thumb is that no more than three references are needed for a given point.  Finally, the last paragraph of the Introduction should briefly summarize the key results (“Tell ‘em what you’re gonna’ to tell ‘em”), and should serve as a transition to the Results section, and it should tie to the first paragraph of the Discussion.

Materials and Methods:

The theoretical goal is that the methods you write out should provide sufficient information for others to repeat your experiments, but this is difficult to do in practice.  Minimize text by referencing previous work and by describing any alterations in the protocol(s) you used.  Consider putting detailed methods and derivations into a Supplemental Methods section.

Statistics:

  • Generally, every symbol in every figure should have an error bar that is defined in the figure legend and in the text. Standard Deviation describes the scatter in the sample, Standard Error of the Mean is used to determine statistical significance.
  • Beware of R-squared, which is a statistical measure of how close the data are to the fitted regression line. It does not denote statistical significance and is inappropriate for nonlinear curve fits.  Consider an F-test.
  • Significant Digits (General Rule of Thumb): Experimental precision limits the significant figures. To allow for later calculations, present uncertainty in a measurement (SD or SEM) with two significant digits and present the mean with one significant digit beyond the largest digit in the uncertainty.  So, 3.4471 +/- 0.238 should be 3.45 +/- 0.24.

Discussion:

The first paragraph of the Discussion should briefly summarize the Results (“Tell em’ what you told ‘em”), and it should set up the entire Discussion that follows.  You should strive to extract as much insight from your data as possible by: (1) making links between different results that you present, (2) connecting your results to published work, and (3) modeling, simulating, or carrying out further analysis of your data, where possible.  You have license to speculate, but it has its limits.  Be sure to note the limitations of your study and your methods.  Be sure to properly cite your colleagues and competitors, and to site all relevant studies that came before.  In the concluding paragraph avoid a generic call for more research, and instead place your work into a larger perspective and relate it to the original questions stated in the Introduction.

Getting feedback

Before submitting your polished manuscript to a journal, give it to lab mates and colleagues and solicit their feedback.  Don’t be defensive in responding to their constructive criticism.  If there are key points that they do not understand, expect reviewers to have the same problems, and work to clarify your message.  Finally, before submitting your manuscript, make sure that pages are numbered.  And good luck with your submission!

References and Resources

S.D. Senturia. How to Avoid the Reviewer’s Axe: One Editor’s View.  J. Micromechanical Systems, 12(3):229–232 (2003).

  • A paper full of sage advice on organizing a paper and persuading your reader.

G.M. Whitesides.  Whitesides’ Group: Writing a Paper. Adv. Materials. 15(16): 1375–1377 (2003).

  • An excellent guide that advocates generating paper outlines early and building them into full manuscripts.

W.A. Wells.  Me Write Pretty One Day:  How to Write a Good Scientific Paper. J. Cell Biol. 165:157–158 (2004).

  • Gives good overview of structuring a paper and developing a narrative.
  1. Spitzer, J. Wildenhain, J. Rappsilber, and M. Tyers. BoxPlotR: A Web Tool for Generation of

Box Plots. Nature Methods, 11(2):121–122 (2014).

  • Advocates for using bean and violin plots to show distributions, rather than bar charts with means or box and whiskers plots.

T.L. Weissgerber, N.M. Milic, S.J. Winham, V.D. Garovic.  Beyond Bar and Line Graphs:  Time for a New Data Presentation Paradigm. PLoS Biol, 13(4): e1002128.doi:10.1371/journal.pbio.1002128 (2015).

  • Demonstrates how much information about distributions and outliers is lost when using bar graphs, and suggests alternative approaches.

Navigating peer review and the publication process will be the subject of Part 3, published Friday, March 10.

Interested in learning more about writing a good biophysics article?  Want a chance to ask questions?  Dr. Hancock will present a webinar on this subject today, March 10, at 1:00 PM ET.  Register at http://www.biophysics.org/Education/Webinars

The Science Behind the Image Contest Winners: Group II Intron Ribozyme

The BPS Art of Science Image Contest took place again this year, during the 61st Annual Meeting in New Orleans. The winning image was submitted by Giulia Palermo, a postdoctoral fellow in the group of J. Andrew McCammon at the University of California, San Diego. A team of three scientists composed the image:  Giulia Palermo created the original design, Amelia Palermo (ETH, Zurich) made the handmade painting, and Lorenzo Casalino (SISSA, Trieste) performed digital manipulation on the picture. Giulia Palermo took some time to provide information about the image and the science it represents.

palermo-giulia-61-70469-2

With this picture we would like to send as the main message that Physics and Art try to interpret the beauty of Nature in different ways but there is a natural overlap between these disciplines, which could lead to wonderful discoveries and amazing beauty.

Group II intron ribozyme perform self-splicing reactions. In the picture, two scissors are used to represent this mechanism. What we like about this image is how a handmade painting could capture the fundamental aspects of the mechanistic action of the system. Besides the beauty of handmade painting, we enjoyed our teamwork and, fostered by the passion for this research, we have been motivated to submit this image to the Art of Science Image Contest.

This image has been inspired by the work we have done in the group of Prof. Alessandra Magistrato (SISSA, Trieste), in collaboration with Prof. Ursula Rothlisberger (EPFL), which resulted in the publication of our research in the Journal of American Chemical Society and in the Journal of Chemical Theory and Computation, while other equally exciting results are in preparation for publication. Below, we report details of our publications:

  1. Casalino, G. Palermo, U. Rothlisberger and A. Magistrato. Who Activates the Nucleophile in Ribozyme Catalysis? An Answer from the Splicing Mechanism of Group II Introns. J. Am. Chem. Soc. 2016, 138, 1034.
  1. Casalino, G. Palermo, N. Abdurakhmonova, U. Rothlisberger and A. Magistrato. Development of Site-specific Mg-RNA Force Field Parameters: A Dream or Reality? Guidelines from Combined Molecular Dynamics and Quantum Mechanics Simulations. J. Chem. Theory Comput. 2017, 13, 340–352.

My research exploits advanced computational methods – based on classical and quantum molecular dynamics (MD), novel cryo-electron microscopy (cryo-EM) refinement – and their integration with experiments to unravel the function and improve biological applications of key protein/nucleic acids complexes directly responsible for gene regulation, with important therapeutic applications for cancer treatment and genetic diseases. As a next-generation computational biophysicist, I aim at going beyond the current limits of time scale and system size of biomolecular simulations, unraveling the function of increasingly realistic biological systems of extreme biological importance, contributing in their applications for effective translational research.

The World Health Organization reported that ~8.2 million citizens die each year for cancer, while genetic diseases affect millions of people. As such, the clarification of the fundamental mechanisms responsible of gene expression and of their therapeutic implications is of key urgency to society.  By using advanced computational methods and by their integration with experiments, I seek to unravel the function and improve applications of biological systems of extreme importance. My current interest – as a post-doc in McCammon’s lab at UCSD – is in the clarification of the mechanistic function of the CRISPR-Cas9 system via computational methods. Additionally, I am interested in long non-coding RNA, which regulates gene expression, and in intriguing protein/DNA systems, whose mechanistic function is at the basis of genetic inheritance.